We have been attempting to develop immunotherapy approaches capable of mediating cancer regression in patients with metastatic cancer. Much of our effort has been devoted to the treatment of patients with metastatic melanoma although more recently (as will be discussed below) we are extending these studies to patients with common epithelial malignancies. A variety of approaches have been explored. In the past four years we have treated over 200 patients with metastatic melanoma or renal cancer with varying schedules of the monoclonal antibody, anti-CTLA-4. Objective response rates by RECIST criteria are 15 to 20%. Approximately 15% of patients developed autoimmune colitis and approximately 9% of patients developed autoimmune hypophysitis. Although T regulatory cells constitutively expressed CTLA-4, we did not see a reduction in T regulatory cells in these patients. Rather we saw an increased activation of circulating lymphocytes as demonstrated by increased expression of activation markers including HLADR. These results have led us to explore alternate means for selectively eliminating T regulatory cells including the use of ONTAK (denileukin diftitox) as well as immunotoxins that combine anti-CD25 with either pseudomonas exotoxin or ricin A chain. We saw no decrease in T regulatory cells by the administration of ONTAK and only partial elimination using immunotoxins. Most of our current efforts are involved with the development of adoptive cell transfer (ACT) approaches to the immunotherapy of cancer. ACT involves the infusion of lymphocytes with anti-tumor activity into cancer patients along with appropriate growth factors to stimulate the survival and expansion of the transferred cells in vivo. Following our description of tumor infiltrating lymphocytes (TIL) capable of recognizing tumor antigens, we performed extensive studies that identified over 50 different new cancer antigens or epitopes recognized by human T cells in a variety of cancer types. Because TIL are capable of recognizing antigens in almost 75% of patients with metastatic melanoma and following pre-clinical work in mouse models, we began a series of studies of the adoptive transfer of these cells into patients with metastatic cancer. In the past four years our recent studies have involved a new generation ACT clinical protocols with altered techniques for TIL growth and with profound host lymphodepletion prior to cell transfer using a non-myeloablative preparative regimen consisting of cyclophosphamide and fludarabine. Objective responses by standard RECIST oncologic criteria were seen in 21 of 43 patients (49%) with metastatic melanoma. In a second trial this same preparative chemotherapy was followed by 200cGy whole body irradiation and autologous hematopoietic stem cells and objective responses were seen in 13 of 25 patients (52%). The lymphodepletion appeared to be necessary to reduce T regulatory cells and to eliminate competition for homeostatic cytokines by endogenous lymphocytes. Several findings in these trials were of note. The transferred cells expanded in vivo and persisted in the peripheral blood in many patients, sometimes achieving levels of 75% of all CD8+ T cells at 6 to 12 months after infusion. Persistence of the transferred T cell clonotypes correlated with cancer regression (p=0.001). In accord with this the telomere length of the infused TIL also correlated with cancer regression (p<0.01). In contrast to murine models the tetramer-positive, antigen-reactive administered TIL were of effector phenotype, CD27-,CD28-,CD45RA-,CD62L-,CCR7-,IL7Ralpha-. Reexpression of CD27 in TIL following removal from IL-2 in vitro also correlates with the effectiveness of these cells in vivo. The success of ACT for the treatment of patients with metastatic melanoma has formed a foundation on which to build improvements of this approach. The ability to introduce genes into circulating human lymphocytes provides the flexibility to introduce antigen receptors as well as molecules that can provide the cell with enhanced properties required for effective ACT therapy. Genes encoding T cell receptors (TCR) were isolated from high avidity T cells that recognized cancer antigens and using retroviral or lentiviral vectors could be used to redirect lymphocyte specificity to cancer antigens. Genes encoding high affinity TCR against melanoma antigens, gp100 and MART-1, against a p53 epitope, against NY-ESO-1 and against CEA have been isolated. Chimeric TCR have been constructed that utilize the combining site of antibodies genetically fused to intracellular T cell signaling chains such as CD3-zeta and can redirect the recognition specificity of lymphocytes to cell surface tumor-associated antigens and thus avoid the limitations of MHC restriction imposed by the use of alpha-beta TCRs. We have recently published the first clinical trial to successfully mediate the regression of human cancer by ACT using genetically engineered autologous lymphocytes. These patients received ACT of their autologous normal lymphocytes transduced with genes encoding a MART-1 TCR. TCRs with far greater affinity for the MART-1 melanoma antigen have been identified and are now being evaluated in clinical gene therapy trials. TCRs are now available against a broad array of cancer antigens present on common epithelial cancers and we have recently begun a trial treating patients with epithelial cancers using autologous T cells transduced with a TCR that recognizes a p53 epitope. Other improvements in ACT are being studied. The lymphodepleting preparative regimens can be increased and we are currently in the midst of a clinical trial using 1200cGy TBI plus chemotherapy as a preparative regimen. Alternatively the preparative regimen can be modified to deplete CD4 cells or selectively eliminate T-regulatory cells. Lymphocyte function may be improved using antibodies or genetic approaches that block inhibitory signals on lymphocytes such as CTLA-4, PD-1, or TGF-beta. Increased persistence and function of the transferred cells may be accomplished by the administration of alternate cytokines such as IL-15 or by stimulating the cells in vivo by the administration of a vaccine or by activating host antigen presenting cells with Toll-like receptor agonists